PROJECT SUMMARY/ABSTRACT The mitochondrial oxidative phosphorylation electron transport chain (ETC) is composed of five large membrane protein complexes (CI, CII, CIII2, CIV and CV) and is responsible for the production of the majority of cellular ATP. Consequently, the ETC is essential to bioenergetic metabolism. ETC defects are one of the most commonly diagnosed congenital metabolic defects, with CI deficiencies representing roughly a third of these diagnoses. Although ~50% of patients with CI deficiencies die within the first 2 years of life and only ~25% reach 10 years of age, CI remains the least well mechanistically understood of all the ETC complexes. Furthermore, despite the large medical need, there are currently no effective treatments for CI or other ETC deficiencies. This discrepancy stems in part from an incomplete understanding of the molecular mechanisms of the individual complexes and their higher-order assemblies into supercomplexes (SCs). In mammalian heart mitochondria the majority of CI is found in association with CIII2 and CIV (SC I+III2+IV, the respirasome) or in association with CIII2 (SC I+III2). Recent biochemical and structural work has produced the first atomic- resolution structures of mammalian mitochondrial CI and defined the arrangement of the individual complexes within the respirasome and SC I+III2. However, significant questions remain regarding the function, mechanism and regulation of the ETC complexes and SCs. To address these gaps in our understanding and to develop the basic science that will underpin potential treatment strategies of ETC defects, we will establish two major research directions in my lab. Using detailed biochemical and enzymatic analyses together with single particle cryo-electron microscopy structural characterizations, we will elucidate the mechanisms, functions and regulation of 1) isolated CI and 2) respiratory SCs. To achieve this, we propose to perform systematic functional and structural comparisons of respiratory CI and SCs purified from mammalian mitochondria (from both HeLa cell culture and porcine heart tissue), the a-proteobacteria Paracoccus denitrificans and the fungal model system Neurospora crassa. P. denitrificans is one of the closest living organisms to the ancestral a- proteobacteria that originated mitochondria after the endosymbiotic event. N. crassa is an established, powerful genetic and biochemical system for bioenergetics, for which nonetheless no high-resolution ETC structures are available. Comparing the CI and SCs from these divergent and genetically tractable organisms to their mammalian counterparts will allow us to test several key mechanistic hypotheses in the field and to identify the conserved features of CI and SC mechanism and regulation. This will provide deep insights into the energy-converting mechanism of CI and the physiological roles of SC formation, which will define the scientific foundation needed for the development of therapeutic strategies against CI and further ETC deficiencies.